1. Technical Field to which the Invention Belongs
This invention relates to a process for producing a peptide or a peptide derivative by using a reaction system of transcribing a DNA into an RNA and then translating the RNA produced or a reaction system of translating an RNA in vitro (hereinafter referred to as “in vitro transcription/translation reaction system”) and a kit of protein components which comprises enzymes and factors for the constitution of this reaction system.
2. Description of the Related Art
There have been known cell-free protein synthesis systems derived from Escherichia coli, rabbit reticulocytes, or wheat germ (Current Opinion in Biotechnology 9:534–548 (1998), J. Biotechnology 41:81–90 (1995)). In these cell-free systems, peptides can be synthesized within several hours. Namely, proteins can be synthesized within a short time compared with the case where foreign genes are inserted into host cells and then expressed therein (Proc. Natl. Acad. Sci. USA 94:412–417 (1997), FEBS Letters 414:268–270 (1997)). Moreover, it is recognized or expected that synthesis of proteins in cell-free systems has a number of technical advantages, at least theoretically, over the case of inserting foreign gene into host cells and expressing the same. Namely, use of these cell-free protein synthesis systems makes it possible to produce peptides which would be digested by proteases originating in host cells and peptides showing toxicity on host cells. It is also possible by using these systems to produce peptide derivatives which do not occur in nature by incorporating unnatural amino acid residues into specific positions by using aminoacyl-tRNA charged by the unnatural amino acid residues (Annu. Rev. Biophys. Biomol. Struct. 24:435–462 (1995)), or complexes (polysome displays) composed of mRNA, ribosome and peptide. These polysome displays and utilization thereof are reported by He M. et al., J. Immunological Methods 231 (2000) pp. 105–117, Schaffitzel C., J. Immunological Methods231 (2000) pp. 119–135, Roberts R W., Current Opinion in Chemical Biology 3 (1999) pp. 268–273 and ibid. 9 (1998) pp. 534–548 (in particular, on and after p. 543) and, in addition, described in, e.g., FEBS Lett. 450:105–110 (1999), Proc. Natl. Acad. Sci. USA 95:14130–14135 (1998), and Proc. Natl. Acad. Sci. USA 94:4937–4942 (1997).
Although crude cell extracts per se were employed at the early stage, only unstable reactions could be performed thereby and thus peptides were synthesized only at low yield, namely, from 0.1 to 0.01% of vital cells. Subsequent studies have clarified components contained in extracts which are necessary for gene expression and simultaneously revealed that unnecessary components and inhibitors (for example endogenous nuclease degrading mRNA (RNA 6:1079–1090 (2000)) are contained therein. Thus attempts have been made to eliminate these unnecessary components. However, the conventional method, which comprises using a cell-free extract as a base and eliminating unnecessary components therefrom, suffers from problems that reaction energy is consumed and thus the reaction stops in about 1 hour in protein synthesis when using a batch system. It is pointed out that factors causative of these problems include starvation of nucleotide triphosphates (Biochim. Biophys. Acta. 1293:207–212 (1996), J. Biotechnol. 48:1–8 (1996)), accumulation of small by-products such as triphosphate hydrolyzates formed by endogenous enzymes (Biochemistry 22:346–354 (1983), J. Biol. Chem. 260:15585–15591 (1985)) and energy consumption by factors unnecessary for the transcription/translation reaction (J. Ferment. Bioeng. 84:7–13 (1997), J. Biotechnol. 61:199–208 (1998)).
The problem of the termination of reaction within a short time can be avoided by continuously supplying a substrate in a transcription/translation reaction system for synthesizing a peptide. However, there arises another problem of poor reproducibility in this case too. This problem has been solved by clarifying the presence of a germ ribosome inactivator (tritin) and a translation initiation inhibitor in studies on a system using wheat germ and employing a means of eliminating these substances from germ (Bio Industry Vol. 17, No.5, 20–27 (2000)). However, there still remains another problem that such a system consumes massive energy source wastefully irrespective of translation.
It was considered that these problems encountering in the conventional methods were caused by the presence of various unknown components in cell extracts which were unnecessary in the transcription or translation reaction but could be hardly eliminated completely. From this viewpoint, an attempt was made to synthesize a peptide in vitro by exclusively using enzymes and factors essentially required in the translation (The Journal of Biological Chemistry Vol. 252, No.19, 6889–6894 (1997)). For the DNA-directed synthesis of β-galactosidase in this case, use was exclusively made of, in addition to E. coli ribosomes, the following 33 components purified from E. coli extract as factors and enzymes for the transcription and translation: RNA polymerase, N10-formyltetrahydrofolate Met-tRNAf transformylase, 20 aminoacyl-tRNA synthetases, IF-1, IF-2, IF-3, EF-Tu, EF-G, RF-1 and/or RF-2, CRP, L and Lα. In this study, however, the target product could be obtained only in a trace amount since only poor information about the translation mechanism and insufficient purification techniques were available in those days.
Subsequently, Gonza and his co-workers constructed an in vitro peptide synthesis system from pre-charged aminoacyl-tRNAs (i.e., having activated amino acid attached thereto) and purified translation factors (Biochem. Biophys. Res. Commun. 126:792–798 (1985)). On the other hand, Pavlov and co-workers reconstructed an in vitro translation system using a partially purified aminoacyl-tRNA synthetase mixture with purified translation factors (Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9–16 (1996)). They also constructed a completely purified in vitro translation system using short artificial mRNA (J. Mol. Biol. 273:389–401 (1997)). However, it has never been reported so far as the inventors know that a protein is successfully synthesized from natural mRNA by using a translation system exclusively comprising essential enzymes and factors. In the conventional cell-free peptide synthesis systems and in vitro peptide synthesis systems using cell extracts, moreover, troublesome procedures are needed for isolating and purifying a target peptide product from protein components in the reaction system and, therefore, the target peptide can be obtained only at a poor yield.